MKK3 Human STE Kinase Enzymatic Radiometric [Km ATP] KinaseProfiler LeadHunter Assay - FR

Procedure Summary:

MKK3 (h) is incubated with 25 mM Tris/HCl, pH7.5, 0.2 mM EGTA, 0.5 mM sodium orthovanadate, 0.5 mM ?- glycerophosphate, 0.01% Triton X-100, 1% glycerol, 10 mM DTT, 0.5 mg/mL MBP, 20 ?g/mL unactive SAPK2a(h), 10 mM Magnesium acetate and [?-33P]-ATP (specific activity and concentration as required). The reaction is initiated by the addition of the Mg/ATP mix. After incubation for 120 minutes at room temperature, the reaction is stopped by the addition of phosphoric acid to a concentration of 0.5%. An aliquot of the stopped reaction is spotted onto a filter and washed four times for 4 minutes in 0.425% phosphoric acid and once in ethanol prior to drying and scintillation counting.

Substrate:

20 ?g/mL unactive SAPK2a(h)

Tracer:

33P

ATP Concentration:

[ATP]= 155µM (Km= 176µM )

Incubation:

120 min at Room temperature

Control Inhibitor:

PP1 Analog II. 1NM-PP1

Test Sample Requirements:

Please provide compounds dissolved in 100% DMSO (PBS or HBSS prohibited).
Stock solution should be ≥50× the final assay concentration (a 10 mM stock is also acceptable).
Required volume:
– <60 kinase assays: 120 µL of 50× stock
– ≥60 assays: 420 µL of 50× stock
If you supply 10 mM stocks or powder (pre-weighed), we will apply the appropriate conversion.
For batches of ~50 compounds supplied as solids, the TAT will be extended.

Minimum Order Quantity:

2